Vasopressin Increases Urinary Acidification via V1a Receptors in Collecting Duct Intercalated Cells : Journal of the American Society of Nephrology

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Vasopressin Increases Urinary Acidification via V1a Receptors in Collecting Duct Intercalated Cells

Giesecke, Torsten1,2; Himmerkus, Nina3; Leipziger, Jens4; Bleich, Markus3; Koshimizu, Taka-aki5; Fähling, Michael6; Smorodchenko, Alina1; Shpak, Julia1; Knappe, Carolin1; Isermann, Julian3; Ayasse, Niklas4; Kawahara, Katsumasa7; Schmoranzer, Jan8; Gimber, Niclas8; Paliege, Alexander9; Bachmann, Sebastian1; Mutig, Kerim1,10

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Journal of the American Society of Nephrology 30(6):p 946-961, June 2019. | DOI: 10.1681/ASN.2018080816
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Abstract

Arginine vasopressin (AVP), also known as antidiuretic hormone, induces urinary concentration by renal salt and water reabsorption via activation of the vasopressin V2 receptor (V2R) in the thick ascending limb, distal convoluted tubule, and principal cells (PCs) of the connecting tubule (CNT) and collecting duct (CD).1–5 Clinically, V2R antagonists have been recognized as a promising pharmacologic tool for retardation of chronic kidney disorders, such as polycystic kidney disease and diabetic nephropathy.6–12

Antagonists of another vasopressin receptor, the V1a vasopressin receptor (V1aR), are also emerging in the treatment of patients with CKD,13–15 although renal V1aR distribution and function have been studied less in comparison with V2R.12,16,17 Previous studies suggested V1aR localization in renal vessels, the macula densa (MD), and intercalated cells (ICs) of CNT/CD, but a clear segmental and cellular distribution could not be defined owing to poor specificity of available antibodies.16,18,19 Among many other functions, V1aR signaling has been implicated in renal acid-base handling and renin release.18–20 V1aR-deficient mice exhibit hypotension due to suppression of the renin-angiotensin-aldosterone system (RAAS) and show distal renal tubular acidosis (dRTA), probably due to impaired functioning of ICs.18–20 Renal ICs include the proton-secreting type A intercalated cells (A-ICs) and the bicarbonate-secreting type B intercalated cells (B-ICs) as well as an intermediate form of non-A, non-B ICs.21 Modulation of their activity by AVP has been demonstrated in several models, yet the mediating pathways remained debatable.19,20,22–24 Elucidation of direct V1aR-mediated effects in ICs has been challenging due to parallel AVP-induced stimulation of RAAS,18,25 complex paracrine interactions between ICs and PCs,26 and uncertainty regarding basolateral versus luminal AVP action.27

In view of increasing availability of highly selective V1aR antagonists and growing interest in their therapeutic potential in the treatment of chronic kidney disorders, it is necessary to substantiate current knowledge on renal V1aR distribution and function. The aim of our study is to provide detailed information on segmental and cellular V1aR distribution in rodent and human kidneys, with a particular focus on its role in renal acid-base handling. Using in vivo and ex vivo models, we address the hypothesis that V1aR activation contributes to urinary acidification via A-ICs.

Methods

An extended methods description is provided in Supplemental Material.

Animal Experiments

All animal experiments were approved by the German or Danish Animal Welfare Regulation Authority and performed in accordance with the European Union Directive 2010/63/EU on the protection of animals used for scientific purposes. For localization studies, adult (8–12 weeks old) male C57BL/6J mice, V1aR-deficient mice, Wistar rats, and AVP-deficient Brattleboro rats with central diabetes insipidus (DI) were euthanized by in vivo perfusion with 3% paraformaldehyde (PFA) in PBS, and kidneys were processed for immunohistochemistry or immunofluorescence (n=6 animals in each group). Physiologic studies with DI rats (n=16) were performed in metabolic cages with water and chow ad libitum. After 2 hours of adaptation, DI rats were treated with the V1aR agonist (A0–4-67)28,29 or vehicle (0.9% NaCl; n=8 each group) for 4 hours, and urine samples were collected hourly under mineral oil. After recovery for 3 days in normal cages, the treatment groups were inverted; the vehicle-treated animals received the agonist, whereas the agonist-treated rats received vehicle using the same experimental protocol. Effects of three agonist doses (200 ng/kg, 2 μg/kg, and 10 μg/kg body weight intraperitoneally) were tested in this way, with recovery periods of at least 3 days between the experimental settings. Finally, DI rats were treated with vehicle (n=6) or the V1aR agonist (2 μg/kg body weight; n=7) for 2 hours and decapitated under isoflurane anesthesia to collect blood samples. The pH and HCO3 in plasma and urine were measured using an ABL 800 Flex analyzer. Net acid excretion (NAE) was determined by titration with the same experimental setting as described previously.30 Deviating from the original protocol, the method was validated for a reduced urine volume of 500 μl per sample (Supplemental Tables 1–4).

Male adult C57BL/6J (n=13) were anesthetized using a mix of ketamine (10 mg/ml) and xylazine (1 mg/ml). The urinary bladder was catheterized, and a micro-pH electrode was placed in the outflow of the catheter to measure urine pH every 5 seconds. After establishing baseline pH values for 30 minutes, mice received the V1aR agonist (AO-4–67; 2 μg/kg body weight intraperitoneally; n=6) or vehicle (0.9% saline intraperitoneally; n=7), and pH was measured for another 60 minutes. The effects of a V1aR antagonist (CL-14–10231,32; 2 mg/kg body weight intraperitoneally; n=3) versus vehicle (0.9% saline; n=3) were assessed in bladder-catheterized male adult C57BL/6J mice fed with a metabolic acidosis–inducing diet (0.28 M NH4Cl in chow plus 0.5% sucrose in water; n=10) for 3 days before the evaluation.33 Morphologic evaluation of V1aR distribution was studied in a parallel cohort of mice receiving regular (n=4) versus the acidosis-inducing diet for 3 days (n=4); animals were euthanized by in vivo perfusion.

Generation of the Anti-V1aR Antibody

The peptide sequence NH2-CKDSPKSSKSIRFIPVST-COOH from the C-terminal mouse V1aR portion was chosen to generate the anti-V1aR antibody due to its negligibly low homology with the vasopressin V2 and V1b receptors and high conservation between the mouse, rat, and human species. Peptide synthesis, immunization of rabbits, and affinity purification of anti-V1aR antibodies were performed by Pineda Antibody-Service (Berlin, Germany). Specificity tests were performed using kidneys from V1aR-deficient mice or human embryonic kidney (HEK293) cells transfected with V1aR or V1bR.

Cell Culture

HEK293 cells were cultured in DMEM medium on coverslips, transfected with GFP-tagged V1aR or control GFP-containing plasmids (pEFGP-N1) using JetPEI transfection reagent, fixed with 3% PFA/PBS for 10 minutes, and processed for double labeling of V1aR and GFP using immunofluorescence. Alternatively, cells were grown in petri dishes and transfected with V1aR, FLAG-tagged vasopressin V1b receptor (V1bR), or control pcDNA3.1 plasmid.34 Cell lysates were precipitated using protein G Sepharose gel (GE Healthcare Life Sciences) and eluates processed for immunoblotting using anti-V1aR or anti-FLAG antibodies (Sigma-Aldrich). Primary inner medullary collecting duct (IMCD) cells were obtained from adult male Wistar rat kidneys using dissection and chemical digestion of renal inner medulla in identical fashion as described previously.35 Cells were grown on permeable filter support systems to full confluence, treated with the V1aR agonist (1.3 μM) or vehicle from the basolateral side for 4 hours, fixed with 4% PFA/PBS, and processed for immunofluorescence.

RT-PCR

mRNA was extracted from microdissected nephron segments using an RNA extraction kit (Stratec Biomedical), and cDNA was synthesized by reverse transcription (Tetro Reverse Transcription; Promega). V1aR-specific forward (5′-CAA TGT CCG AGG GAA GAC AG-3′) and reverse primers (5′-GTT GGG CTT CGG TTG TTA GA-3′) were designed, and RT-PCR was performed in an automated thermal cycler (PerkinElmer) using Taq polymerase (GIBCO).

Immunofluorescence, Immunohistochemistry, and Quantitative Analyses

Paraffin-embedded kidney sections (4 μm) were dewaxed, boiled in citrate buffer (pH 6, 6 minutes), washed in TBS, and blocked with 5% skim milk in TBS for 30 minutes. Cultured cells on coverslips were permeabilized for 10 minutes using 0.5% Triton X-100/TBS and blocked with 5% BSA in TBS for 30 minutes. Primary antibodies to V1aR (own antibody), aquaporin 2 (AQP2; sc-9882; Santa Cruz Biotechnology, Dallas, TX), pendrin (a gift from C.A. Wagner, Zurich, Switzerland), vacuolar H+-ATPase (V-ATPase, sc-20943; Santa Cruz Biotechnology), and GFP (ab291–50; abcam, Cambridge, United Kingdom) were applied for 1 hour at room temperature followed by overnight incubation at +4°C. Primary antibodies were detected using fluorescent Cy2-, Cy3-, or Cy5-conjugated (Dianova, Hamburg, Germany) or HRP-conjugated secondary antibodies (Dako, Glostrup, Denmark). Double and triple staining was performed by sequential application of respective primary and secondary antibodies separated by washing steps. Fluorescent signals were evaluated by confocal microscopy using a Zeiss LSM 5 Exciter microscope with 40× and 63× objectives (N.A. 1.3/1.4) and processed with ZEN 2008 software. Light microscopy images were acquired with a LEICA DMRB microscope using a 100× objective (N.A. 1.30) and processed with the Axio Vision SE64 software. Three-dimensional structured illumination microscopy images were acquired using the OMX V4 Blaze system (GE Healthcare). Quantification of confocal signals by intensity was performed using Fiji software. Quantification of A-ICs, B-ICs, and PCs numbers in CNT/CD was performed in kidney sections concomitantly labeled for AQP2, pendrin, and V-ATPase or V1aR to identify PCs by their luminal AQP2 signal, B-ICs by their luminal pendrin and basolateral V-ATPase labeling, and A-ICs by their luminal V-ATPase staining.

Isolated Perfused Collecting Ducts

Adult (8–12 weeks) male C57BL/6J mice were euthanized by decapitation under isoflurane anesthesia followed by removal of the kidneys. CDs were dissected at the transition zone between cortex and outer medulla and processed for measurements of intracellular calcium concentrations ([Ca2+]i)or luminal pH. Four to six CDs were analyzed in each experimental setting. CDs were perfused with a double-barreled perfusion system of concentric pipettes in a temperate bath chamber. All measurements were performed in a pregassed (95% O2 and 5% CO2) bath solution. For [Ca2+]i measurements, CDs were incubated with 10 μmol/L Fura-2-AM in dissection solution for 1 hour at room temperature, and fluorescence intensities at 340 and 380 nm were monitored using an inverted microscope. After obtaining baseline values, CDs were treated with the V1aR agonist (A0–4-67; 50 or 100 nM) for 3 minutes followed by a washout period of 7–8 minutes and application of 50 nM AVP for 3 minutes; 340-to-380-nm signal ratios were calculated as an indicator of [Ca2+]i, and their peaks were compared between the treatments. For assessment of the luminal pH, CDs were perfused with 100 μmol/L 2ʹ,7ʹ-bis(carboxyethyl)-5(6ʹ)-carboxyfluorescein in luminal solution, and intensities of luminal fluorescence at 486 and 440 nm were monitored to calculate 486-to-440-nm signal ratio as a pH indicator. After equilibration for 5–10 minutes, CDs were treated with the V1aR agonist (100 nM for 4 minutes in the bath solution) followed by a washout for 10 minutes and application of 50 nM AVP for 4 minutes. Effects of the V1aR agonist and AVP on luminal pH were compared after normalization to the mean baseline values obtained during 30 seconds before the respective treatments.

Statistical Analyses

Data are presented as means and SEM. We assumed normal distribution of our results on the basis of the experimental design. Results of animal experiments were evaluated by unpaired two-tailed t test to determine statistical significance. Ex vivo settings were analyzed by unpaired or paired t test and Kruskal–Wallis test followed by Dunn multiple comparisons test; P<0.05 was accepted as a statistically significant difference.

Results

Segmental and Cellular V1aR Distribution in Rodent and Human Kidneys

To study the renal distribution of V1aR, we generated a polyclonal antibody to this receptor subtype by immunizing rabbits with a synthetic peptide corresponding to a C-terminal mouse V1aR portion (NH2-CKDSPKSSKSIRFIPVST-CONH2) with no significant homology to mouse V1bR or V2R but substantial homology to rat and human V1aR. Immunoperoxidase staining of mouse kidney produced basolateral or perinuclear to apical V1aR signal patterns in ICs of CNT and CD segments (Figure 1, A and B). Triple immunofluorescence labeling of V1aR, AQP2 as the luminal marker for PCs, and pendrin as the luminal marker for B-ICs allowed us to assign the basolateral V1aR signal to A-ICs and the perinuclear/subapical V1aR signal to B-ICs displaying apical pendrin (Figure 1, C–F; Supplemental Figure 1). Apical pendrin staining did not coincide with the V1aR signal, suggesting that the receptor did not reach the luminal membrane in B-ICs (Figure 1F). The distinct V1aR distribution patterns in A-ICs versus B-ICs were further verified using high-resolution three-dimensional structured illumination microscopy imaging (Figure 1, G and H; Supplemental Movie). In a small proportion of pendrin-positive cells, the V1aR signal was absent or showed either basolateral or diffuse localization (Figure 2, A–C). Similar to mice, rat and human kidneys showed basolateral or intracellular to subapical V1aR signals in AQP2-negative ICs (Figure 2, D–G). These localization data suggest basolateral V1aR-mediated effects of AVP in A-ICs, whereas B-ICs may be less responsive to AVP considering the virtual absence of V1aR in their plasma membrane. Other sites displaying renal V1aR immunoreactivity were MD cells showing a clear basolateral signal in mouse but not in rat or human kidney samples (Supplemental Figure 2). Renal arterioles and vasa recta showed a V1aR immunoreactive signal in the endothelium (Supplemental Figure 3). To further corroborate the localization data, we evaluated V1aR mRNA expression in microdissected mouse nephron segments using RT-PCR, which produced specific signals in CNT/CD and glomeruli with attached MD but not in proximal tubules, thick ascending limb, or distal convoluted tubule (Figure 1I, Supplemental Figure 4).

fig1
Figure 1.:
Vasopressin V1a receptor (V1aR) is distinctly distributed in intercalated cells (ICs) of connecting tubule and collecting duct segments in mouse kidney. (A and B) Immunoperoxidase staining shows vasopressin V1 receptor signal in intercalated cells (ICs) of connecting tubule; signal is located either on the (A) basolateral or (B) luminal side; bright-field microscopy and differential interference contrast optics. (C–F) Triple immunofluorescence staining shows V1aR signal (green), aquaporin 2 (AQP2) signal (magenta) and pendrin signal (red) in cortical collecting duct; merged images are shown. Apical V1aR signal is coexpressed with pendrin in bicarbonate-secreting type B intercalated cells (B-ICs; closed arrows), whereas basolateral V1aR signal is present in AQP2- and pendrin-negative proton-secreting type A intercalated cells (A-ICs; thin arrows; inset magnified in E). In detail, subapical pendrin overlaps with V1aR signals, whereas luminal pendrin does not (merged orange versus red or green signals; inset magnified in F). (G and H) Structured illumination microscopy shows V1aR immunoreactivity of cortical collecting duct ICs at high resolution; signals are distributed (G) along the basolateral membrane of an A-IC or (H) in the cytoplasm with subapical accumulation in a B-ICs (Supplemental Movie). Basement membranes are indicated by dashed lines, and apical cell membranes are indicated by dotted lines. Nuclear counterstaining is with 4′,6-diamidino-2-phenylindole (blue). (I) RT-PCR analysis of V1aR expression in microdissected mouse nephron segments shows V1aR signals in glomeruli (Glom.) with attached macula densa (Glom.+MD), connecting tubules and cortical collecting ducts (CNTs/CDs) and medullary collecting ducts (mCD) but not in proximal convoluted segments (PCTs), proximal straight segments (PSTs), or thick ascending limbs (TALs). The weak signal in the distal convoluted tubule (DCT) is likely due to ICs of late DCT and attached initial CNT portion (DCT); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) detection serves as the loading control.
fig2
Figure 2.:
Vasopressin V1a receptor (V1aR) exhibits similar expression patterns across the mouse, rat and human species and shows variable distribution in pendrin-positive intercalated cells (ICs). (A and B) Images of mouse connecting tubule/cortical collecting duct showing one IC with basolateral V1aR (green) and apical pendrin staining (red; arrow) and another with no V1aR signal and apical pendrin staining in A (arrowhead) and two ICs with basolateral and perinuclear V1aR and concomitant luminal pendrin signals in B (arrows). Adjacent principal cells display luminal aquaporin 2 (AQP2) staining (magenta). (C) Schematic diagram summarizing the pleomorphism of distinct V1aR distribution patterns in pendrin-positive compared with pendrin-negative ICs with reference to the individual images shown here and in Figure 1. (D and E) ICs with basal V1aR signal (green) in human kidney medullary collecting duct (thin arrows in D) and luminal V1aR signal in cortical collecting duct (closed arrows in E). (F and G) Rat renal cortical collecting ducts showing pendrin-negative ICs with basolateral V1aR signal (thin arrows in F and G) or pendrin-positive ICs with mild to absent intracellular or subapical V1aR signal (closed arrows in F and G). (D–G) Adjacent principal cells display luminal AQP2 staining.

Specificity of the anti-V1aR antibody was confirmed by labeling of V1aR-deficient kidneys, which produced no significant signal in ICs or MD cells (Figure 3, A–D; Supplemental Figure 2, C and D). Immunoblotting with this antibody showed abundant signal of the expected molecular weight range in mouse liver samples, whereas brain and kidney signals were weak, possibly reflecting distinct numbers of V1aR-expressing cells in these organs (Figure 3E). Additional specificity tests used transfection experiments in cultured HEK293 cells lacking endogenous V1aR (Figure 3F). Transfection of mouse V1aR, FLAG-V1bR, or empty pcDNA3.1 plasmid followed by precipitation with protein G Sepharose gel and immunoblotting using anti-V1aR or anti-FLAG antibodies confirmed specificity of our antibody to V1aR and lack of crossreactivity with V1bR (Figure 3G). Along the same line, immunofluorescence labeling of HEK293 cells transfected with V1aR-GFP or control GFP-containing plasmid produced a clear plasma membrane–associated V1aR signal in the V1aR-GFP–transfected cells but not in controls (Figure 3, H–K).

fig3
Figure 3.:
Verification tests with the anti-vasopressin V1a receptor (anti-V1aR) antibody confirm its specificity. (A–D) Representative immunofluorescence images of cortical collecting duct in wild-type (WT) versus vasopressin V1a receptor knockout (V1aR-KO) kidney sections triple labeled for V1aR (green signal), aquaporin 2 (AQP2; magenta signal), and pendrin (red signal) show basolateral V1aR signal in AQP2- and pendrin-negative proton-secreting type A intercalated cells (A-ICs; thin arrows) along with intracellular V1aR signal in pendrin-positive bicarbonate-secreting type B intercalated cells (B-ICs; closed arrows) in (A and C) WT but not in (B and D) V1aR-KO mice. (E) Representative immunoblot of mouse kidney, liver, and brain lysates using anti-V1aR antibody shows strong signal in liver samples and weaker signals in brain and kidney samples (all at approximately 47 kD). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serves as the loading control (three independent experiments). (F) RT-PCR using specific primers for V1aR mRNA detects a V1aR product of expected size (460 bp) in WT mouse kidney cDNA but not in cDNA obtained from human embryonic kidney (HEK293) cells. (G) Representative immunoblots detecting V1aR or FLAG in eluates obtained by G protein sepharose precipitation of lysates from HEK293 cells transfected with empty pCDNA3.1 plasmid (Vector), mouse vasopressin V1a receptor (mV1aR), or FLAG-tagged mouse vasopressin V1b receptor (FLAG-mV1bR). Specific V1aR signal was obtained only in samples from mV1aR-transfected cells but not from FLAG-V1bR–transfected cells, suggesting the lack of crossreactive V1bR recognition by our antibody (three independent experiments). (H–K) Representative confocal microscopic images of cultured HEK293 cells, transfected with green fluorescent protein (GFP) tagged human V1aR plasmid (V1aR-GFP) or GFP control plasmid, show membrane associated V1aR signal in cells expressing V1aR-GFP (H; red signal) as identified by concomitant GFP fluorescence (J; green signal), but no significant V1aR signal in cells transfected with the GFP control plasmid (I, K).

Effects of V1aR Deletion on Distribution of CNT/CD Cell Types in Mouse Kidney

To test whether V1aR signaling affects the numerical proportions of IC types in CNT and CD, we quantified the numbers of PCs, A-ICs, and B-ICs in cortex and medulla of wild-type versus V1aR-deficient kidneys using triple labeling for AQP2, V-ATPase, and pendrin. The two genotypes showed similar percentages of PCs, A-ICs, and B-ICs, suggesting that V1aR is not essential for their proportional distribution in CNT and CD (Table 1).

Table 1. - Distribution of collecting duct cell types in wild-type versus vasopressin V1a receptor–deficient kidneys.
Category Cortex, % Medulla, %
PCs A-ICs B-ICs Outer Medulla Inner Medulla
Outer Stripe Inner Stripe PCs A-ICs
PCs A-ICs PCs A-ICs
WT 55.59 18.22 26.19 75.92 24.08 76.28 23.72 89.69 10.31
V1aR-KO 55.39 17.23 27.38 71.77 28.23 72.84 27.16 90.41 9.59
Δ −0.20 −0.99 1.19 −4.15 4.15 −3.44 3.44 0.72 −0.72
P value 0.92 0.69 0.72 0.15 0.15 0.23 0.23 0.71 0.71
Triple labeling for aquaporin 2 (AQP2), V-ATPase, and pendrin was performed to differentiate between the AQP2-positive principal cells (PCs), AQP2- and pendrin-negative type A intercalated cells (A-ICs), and pendrin-positive type B intercalated cells (B-ICs) in wild-type (WT; n=4) versus vasopressin V1a receptor–deficient (V1aR-KO) kidneys (n=4). At least ten to 20 tubules were quantified in each kidney zone; data are means.

Functional Effect of V1aR Stimulation versus Inhibition In Vivo

To mimic the physiologic effect of AVP binding to V1aR, we treated anesthetized and urinary bladder–catheterized mice with the V1aR agonist, AO-4–67 (2 μg/kg body weight), or vehicle. Compared with baseline values, urinary pH was significantly decreased after 20 minutes of the agonist administration (pH from 7.18 to 6.78; P<0.05) (Figure 4A) but unaffected in controls, indicating that selective stimulation of V1aR increases urinary acid load in vivo. To corroborate these results, we took advantage of AVP-deficient Brattleboro rats with central DI. Analysis of V1aR distribution in DI rats produced a signal pattern similar to that of normal rats (i.e., basolateral receptor presence in A-ICs versus diffused intracellular distribution in B-ICs) (Supplemental Figure 5, A–D). DI rats were treated with the V1aR agonist (200 ng/kg, 2 μg/kg, or 10 μg/kg body weight for 4 hours) to study changes of NAE in metabolic cages. The highest dose reduced urinary pH (from 7.40 to 6.71, 6.71, 6.56, and 6.53 after 1, 2, 3, and 4 hours, respectively; P<0.01) and HCO3 levels (−82%, −92%, and −85% after 2, 3, and 4 hours, respectively; P<0.001). NH4+ excretion was not significantly altered, but NAE showed significant increases (+176%, +248%, and +98% after 2, 3, and 4 hours, respectively) (Figure 4, C–F). The lower agonist doses produced only minor changes in the urine but led to a significant increase in plasma HCO3 without concomitant change of pH, likely reflecting the spared bicarbonate (27.6 versus 29.2 mmol/L; P<0.01) (Supplemental Figure 5E).

fig4
Figure 4.:
Pharmacologic stimulation of vasopressin V1a receptor (V1aR) decreases urinary pH and promotes urinary net acid excretion (NAE) in rodents. (A) Time course of urinary pH in bladder-catheterized mice after single intraperitoneal injections of 0.9% saline (vehicle; n=7) or V1aR agonist A0–4-67 (2 μg/kg body weight; n=6); the arrow indicates the time point of saline or agonist application. (B) Time course of urinary pH in acid-loaded (0.28 M NH4Cl for 3 days) bladder-catheterized mice after single intraperitoneal injections of 0.9% saline (vehicle; n=3) or V1aR antagonist (CL-14–102; 2 mg/kg body weight; n=3); the arrow indicates the time point of saline or antagonist application. Data are means ± SEM; *P<0.05 over the time interval indicated by the black line; n.s., not significant. (C–F) Time course of (C) urinary pH, (D) HCO3 excretion, (E) net acid excretion (NAE), and (F) NH4 + excretion in Brattleboro rats housed in metabolic cages and treated with single intraperitoneal injections of 0.9% saline (vehicle; n=16) or A0–4-67 (200 ng/kg, 2 μg/kg, or 10 μg/kg body weight; each n=16). Urine was collected hourly. Data are means ± SEM. *P<0.05 versus vehicle; **P<0.01 versus vehicle; ***P<0.001 versus vehicle.

To study the role of V1aR during increased acid load, we induced metabolic acidosis in mice by administering 0.28 M NH4Cl with chow for 3 days.33 The resulting metabolic acidosis was confirmed by changes in plasma pH, HCO3, and Cl levels (Supplemental Figure 6). Intracellular V1aR distribution and proportions of V1aR-expressing ICs were unaffected in acidotic mice (Supplemental Figure 7, Supplemental Tables 1–4). Acute application of a V1aR antagonist in acidotic, bladder-catheterized mice (CL-14–102; 2 mg/kg body weight intraperitoneally) produced no significant changes in urinary pH compared with vehicle treatment (Figure 4B).

Effects of V1aR Stimulation on V-ATPase in Primary Cell Culture

To address V1aR-mediated effects on urinary acidification, IMCD cells isolated from rat kidney papilla were studied.35 To this end, V-ATPase expression in V1aR-immunoreactive A-ICs was first identified in situ in their papillary environment using double labeling for AQP2 and V1aR or for AQP2 and V-ATPase; A-ICs revealed substantial luminal V-ATPase and basolateral V1aR signals as opposed to AQP2-positive PCs (Figure 5, A and B). B-ICs were typically absent from this kidney zone. Isolated IMCD cells grown to confluence on permeable support similarly expressed V1aR and V-ATPase in single A-ICs scattered among AQP2-positive PCs (Figure 5, C and D). V1aR staining was detected along the lateral cell borders, whereas V-ATPase staining was distributed over the entire cell. To evaluate the effects of V1aR stimulation on V-ATPase in A-ICs, IMCD cells were treated with the V1aR agonist (1 μM, 4 hours) or vehicle applied from the basolateral side. V-ATPase immunoreactive signal intensity was measured using confocal z stacks of individual A-ICs over 7.5 μm of apicobasal extension. Apical V-ATPase signal was substantially enhanced in the supranuclear region of cells receiving the agonist as demonstrated by intensity plotting (+93%; P<0.001) (Figure 6, A–F). These data suggest vasopressin-inducible, V1aR-mediated activation of V-ATPase in A-ICs.

fig5
Figure 5.:
Primary cell culture of rat inner medullary collecting duct (IMCD) cells contains type A intercalated cells (A-ICs), expressing vasopressin V1a receptor (V1aR) and vacuolar H+-ATPase (V-ATPase). (A and B) Inner medulla of perfusion-fixed rat kidney shows luminal aquaporin 2 (AQP2) staining in principal cells (green) and (A) vasopressin V1a receptor (V1aR) or (B) V-ATPase staining (red) in AQP2-negative proton-secreting type A intercalated cells (A-ICs) in situ. (C and D) Rat primary IMCD cells grown to confluence on permeable support show two cell populations: principal cells (PCs) stained for AQP2 (green) and A-ICs stained for (C) V1aR (red) or (D) V-ATPase (red). Signals are membrane associated (V1aR) or cytoplasmic (V-ATPase). Nuclear counterstaining was with 4′,6-diamidino-2-phenylindole (blue).
fig6
Figure 6.:
Basolateral stimulation of vasopressin V1a receptor (V1aR) in cultured inner medullary collecting duct (IMCD) cells increases luminal abundance of vacuolar H+-ATPase (V-ATPase) in type A intercalated cells (A-ICs). (A–D) Representative confocal images of confluent IMCD cells treated from the basolateral side with (A and C) vehicle or (B and D) V1aR agonist (A0–4-67; 1 μM for 4 hours) and labeled for V-ATPase (red). Cellular detail is shown by (C and D) confocal stacks from selected cells as indicated by (A and B) arrows; nuclear counterstaining was with 4′,6-diamidino-2-phenylindole (blue). Increased V-ATPase signal intensity and selective apical enhancement characterize the effects of the agonist. (E) Distribution of apical V-ATPase signal intensities across two representative intercalated cells: one treated with vehicle and the other treated with agonist (confocal z stacks). §Dashed line indicates the distance of 7.5 μm from the luminal membrane. (F) Quantification of apical V-ATPase signal intensities (as in E) in cultured vehicle- (n=202) or agonist-treated intercalated cells (n=151) obtained from three independent experiments including three animals each. Box and whisker plots show medians and interquartile ranges in the boxes. The whiskers are quartile 1 + interquartile range ×1.5 or quartile 3 + interquartile range ×1.5; black dots represent data from individual intercalated cells. ***P<0.001 for vehicle versus agonist.

Effects of V1aR Stimulation in Isolated Perfused Collecting Duct Segments

Cellular V1aR signaling depends on intracellular Ca2+ release. We, therefore, used isolated perfused outer medullary CDs from mouse kidney to measure [Ca2+]i and luminal pH using Ca2+- (Fura-2 AM) and pH-sensitive [2ʹ,7ʹ-bis(carboxyethyl)-5(6ʹ)-carboxyfluorescein] fluorescent dyes.36,37 Basolateral treatment of CDs with the V1aR agonist, A0–4-67, induced dose-dependent, transient increases of [Ca2+]i. The consecutive application of AVP exerted stronger effects compared with a low V1aR agonist dose (1.20-fold for 50 nM A0–4-67 versus 1.50-fold for 50 nM AVP; P<0.05) but was comparable with a higher dose of the V1aR agonist (1.40-fold for 100 nM A0–4-67 versus 1.45-fold for consecutive 50 nM AVP) (Figure 7, A–E). We, therefore, selected the higher agonist dose to evaluate effects on luminal pH. Here, basolateral application of A0–4-67 or AVP caused significant luminal acidification to a similar extent (−4.55% for 100 nM A0–4-67 versus −4.15% for consecutive 50 nM AVP; P<0.05 for each treatment versus respective baseline values) (Figure 7, F and G). Together, our data indicate that V1aR stimulation induces luminal H+ secretion in CDs, thus corroborating and extending the in vivo data.

fig7
Figure 7.:
Basolateral application of arginine-vasopressin (AVP) or a V1aR agonist (A0-4-67) increases intracellular calcium levels and reduces luminal pH in isolated and perfused mouse collecting ducts (CDs). (A–C) Representative transmission light microscopic images showing (A) an isolated perfused CD, (B) its fluorescent image at 340-nm excitation, and (C) the respective 340-to-380-nm signal ratio in a Fura-2-AM–loaded CD for intracellular calcium concentration ([Ca2+]i) detection. (B) The evaluated regions are labeled with circles. (D) Graphs summarizing changes of the 340-to-380-nm signal ratio in response to application of the V1aR agonist A0–4-67 (indicated by §; 50 or 100 nM) followed by washout (indicated by #) and consecutive application of arginine vasopressin (AVP; indicated by &; 50 nM). (E) Diagrams showing the changes for the 340-to-380-nm signal ratios, reflecting changes of [Ca2+]i in response to the two V1aR agonist doses (50 [I] or 100 nM [II]) and AVP (50 nM) after washout compared with the respective baseline levels obtained before treatment (n=4 CDs per group; three independent experiments). *P<0.05 compared with baseline values; # P<0.05 for comparison of strength of effects of V1aR agonist versus AVP application. (F) Changes of luminal pH in 2ʹ,7ʹ-bis(carboxyethyl)-5(6ʹ)-carboxyfluorescein–perfused CDs depicted as the 486-to-440-nm signal ratio in response to application of the V1aR agonist A0–4-67 (indicated by §; 100 nM, n=7) followed by washout (indicated by #) and consecutive application of AVP (indicated by &; 50 nM, n=6; four independent experiments). Representative fluorescent image (486 nm) showing the isolated perfused CD with the evaluated region (circle). (G) Diagrams showing the changes in luminal pH as reflected by fluctuations of the relative 486-to-440-nm signal ratios (comparison of pre- and postcontrol) in response to the V1aR agonist (100 nM [III]) and consecutive AVP (50 nM [III]) or in response to AVP treatment only (IV) without preceding A0–4-67 application compared with respective baseline levels obtained before treatments. Data are the means ± SD. *P<0.05.

Discussion

In this study, we focused on the role of ICs in AVP-V1aR–mediated control of acid-base homeostasis in the CD system. Expression of V1aR in CNTs and CDs has been reported, yet cell type–specific characterization of its distribution remained uncertain.16,19,20,38,39 Earlier localization and functional studies suggested predominantly apical V1aR distribution in ICs or PCs.19,39–43 Indeed, AVP is filtered into the urine, where its concentration substantially exceeds plasma levels.5,27 Luminal effects of AVP on [Ca2+]i or transepithelial resistance have been established in the rabbit CD, whereas robust functional data from rodent or human kidneys are not available.40,43 Other studies demonstrated basolateral effects of V1aR stimulation in rabbits and rats, although the responsive cell types were not defined.41,44

In this work, we used a new anti-V1aR antibody to clarify segment- and cell type–specific aspects of V1aR distribution in mouse, rat, and human kidneys. The clear basolateral, membrane-bound V1aR signal in A-IC is suggestive of their responsiveness to plasma AVP, whereas the intracellular signal without significant plasma membrane association in B-ICs points to their poor AVP sensitivity compared with A-ICs.

Our functional studies further support direct effects of AVP in A-ICs. Stimulation of the AVP-V1aR axis in vivo by a V1aR agonist decreased urinary pH and increased NAE in AVP-deficient DI rats, which is consistent with activation of A-ICs. Because of the lack of endogenous AVP, DI rats were assumed to exhibit particularly strong responses to AVP receptor agonists.45 However, a relatively high dose of V1aR agonist was required to induce significant effects in this model, which may be related to altered urinary buffering capacities in the state of extreme diuresis or volume depletion in Brattleboro rats. V1aR activation may further elicit indirect effects via potentiation of aldosterone action.19 Bladder catheterization in mice has permitted particularly robust recordings of the urinary acid-base status.37 To over-ride the effects of endogenous AVP, we applied a saturating dose of V1aR agonist in mice, which resulted in rapid decreases of urinary pH, likely due to activation of A-ICs.

These results were extended in our ex vivo models using isolated perfused CDs or cultured IMCD cells. Because V1aR signals via intracellular calcium release,46 we have detected [Ca2+]i parallel to evaluation of luminal pH in isolated CDs. Basolateral application of the agonist in isolated perfused CDs reduced luminal pH and elicited transient [Ca2+]i increases, which is in line with the assumed recruitment of intracellular calcium as a second messenger.47 Because virtually all epithelial cells of isolated tubules exhibited increased [Ca2+]i in response to the agonist, we suggest that direct effects on A-ICs may elicit paracrine signaling events affecting neighboring PCs.21 Administration of AVP produced similar increases of [Ca2+]i and luminal pH reduction as the V1aR agonist, suggesting that urinary acidification is not an artifact of selective V1aR stimulation but rather, a physiologic AVP function. These results also suggest that concomitant V2R activation in PCs does not elicit inhibiting paracrine effects on V1aR-mediated H+ secretion. The model of isolated perfused CDs thus permitted us to detect significant effects of basolateral V1aR activation on luminal pH in the absence of systemic factors or contribution of upstream nephron segments. As to mechanisms involved, our experiments in cultured IMCD cells suggest stimulation of V-ATPase via its luminal trafficking.48 Cultured IMCD cells have been previously established as a model of PCs function.35 Here, we made use of these cells to study regulation of A-ICs, which are present in IMCD along with PCs.21,49 Growing IMCD cells on permeable support enabled basolateral application of the V1aR agonist. The resulting signal increases of apical V-ATPase in A-ICs suggested activation of the proton pump via luminal trafficking, although we cannot exclude changes in biosynthesis rate or protein stability.48 Other H+-secreting proteins may be affected in parallel.50 Previous work showed that aldosterone also activates V-ATPase in A-ICs via its luminal trafficking in a Ca2+-dependent manner, which may reflect involvement of a common downstream mediator, such as protein kinase C.51

V1aR-deficient mice exhibit dRTA,19,52 which led us to test the role of V1aR in renal adaptation to acid load using an NH4Cl-rich diet.33 Previous work reported increased V1aR mRNA expression in ICs of acid-loaded mice.20 Our data showed unaltered cellular V1aR distribution upon acid loading, suggesting an absence of compensatory changes at this level. V1aR antagonist had no effects on urinary pH in this model, suggesting a minor role for V1aR signaling in adaptations to metabolic acidosis. Local pH-dependent modulation of V1aR signaling in ICs may be considered as well (i.e., H+ secretion may be stimulated by local alkaline pH but inhibited by acidic pH).53

The nondetectable membrane-associated V1aR signal in B-ICs, despite intracellular receptor accumulation, suggests that AVP does not elicit direct effects in this cell type. However, minor luminal surface expression of the receptor below the given antibody detection limit may still be functional considering the naturally high urinary concentrations of AVP.5,27 In this case, rapid binding of AVP to luminal V1aR followed by receptor internalization could potentially explain its intracellular accumulation in B-ICs.5,54,55 However, this was not confirmed in AVP-deficient DI rats, which also lacked a membrane-bound receptor signal.

In this context, neither V1aR deletion in mice nor V1aR knockdown in cultured ICs altered the expression of pendrin, the Cl/HCO3 exchanger of B-ICs.19 In contrast, in V2R-deficient mice, pendrin was suppressed in these cells.56 Along the same line, the V2R agonist desmopressin increased renal pendrin expression in DI rats.23 Because V2R is not expressed in ICs,57 these effects may have been elicited by neighboring V2R-expressing PCs via paracrine interactions involving PG, purinergic signaling, or other pathways.21,26,58,59 Available data thus do not provide evidence for a direct sensitivity of B-ICs to AVP but suggest paracrine crosstalk between different CD cell types.40,42,60

Mechanisms underlying the distinct V1aR distribution patterns in A-ICs and B-ICs are not clear at present. A recent transcriptome analysis demonstrated similar abundances of V1aR mRNA expression in both cell types, suggesting that post-translational regulation determines the individual destination of the receptor.61 Extrinsic factors may potentially modulate functional properties of ICs, including AVP signaling mechanisms; adaptive conversion of B-ICs to A-ICs or proliferation of A-ICs serves to prevent dRTA.26,52,62 The role of A-ICs in the renal acid-base handling is illustrated by pronounced dRTA in hensin-deficient mice lacking this IC-type due to impaired hensin-dependent conversion of B-ICs to A-ICs.63 Although our data suggest functional V1aR-dependent activation of A-ICs, we obtained no evidence for a role of V1aR in IC acquisition of distinct phenotypes.

Apart from direct effects on A-ICs, AVP may affect renal acid-base handling via stimulation of RAAS.18,25,64 RAAS components exert permissive effects on functions in all CD cell types.21,58,65,66 Angiotensin II and aldosterone stimulate V-ATPase in A-ICs, thus increasing luminal H+ secretion, whereas aldosterone additionally induces pendrin in B-ICs, which facilitates the function of the epithelial sodium channel in PCs.65–69 The AVP-dependent modulation of RAAS may take place at the level of hypothalamus via stimulation of adrenocorticotropic hormone release as reported in rats.70 In mice, effects of AVP on RAAS may additionally be mediated by V1aR in MD cells, because V1aR-deficient mice exhibited hyporeninemia and low BP.18

In summary, this study extends information on V1aR distribution in rodent and human kidneys and provides several lines of evidence for AVP-induced, V1aR-mediated urinary H+ secretion by A-ICs.

Disclosures

None.

Published online ahead of print. Publication date available at www.jasn.org.

We thank Kerstin Riskowsky and Frauke Grams for excellent technical assistance; Martin Thomson (Charité–Universitätsmedizin Berlin), Finn Antrobus and Yvonne Giesecke (both University of St. Andrews) for proofreading the manuscript; Prof. Carsten Wagner (Zurich, Switzerland) for providing us with the antipendrin antibody; Prof. Maurice Manning (Toledo, OH) for providing us with the V1aR agonist A0-4-67 and with the V1aR antagonist CL-14-102; and Yvonne Giesecke for help with cell quantification.

The work was supported by the Charité–Universitätsmedizin Berlin and Deutsche Forschungsgemeinschaft grants MU2924/2-2 and BA700/22-2. Mr. Torsten Giesecke received a joint research grant from the Berlin Institute of Health and the Charité–Universitätsmedizin Berlin. The work of Dr. Gimber was supported by the Advanced Medical BioImaging Core Facility of the Charite–Universitätsmedizin Berlin and Deutsche Forschungsgemeinschaft grant SFB958/Z02 (to Dr. Jan Schmoranzer). Dr. Paliege was supported by the Charité Clinical Scientist Program financed by the Charité–Universitätsmedizin Berlin and the Berlin Institute of Health.

Supplemental Material

This article contains the following supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2018080816/-/DCSupplemental.

Supplemental Figure 1. Overview of V1aR distribution in mouse kidney.

Supplemental Figure 2. Distribution of V1aR in macula densa cells.

Supplemental Figure 3. V1aR distribution in renal vasculature of mouse kidney.

Supplemental Figure 4. Evaluation of microdissected nephron segments.

Supplemental Figure 5. V1aR distribution in Brattleboro rats and effects of the V1aR agonist.

Supplemental Figure 6. Verification of metabolic acidosis in mice.

Supplemental Figure 7. Effects of metabolic acidosis on cellular V1aR distribution.

Supplemental Material. Complete methods: extended methods description.

Supplemental Movie. 3D reconstruction of structured illumination microscopy (3D-SIM) showing subcellular distribution of V1aR in mouse kidney.

Supplemental Table 1. Proportion of collecting duct cell types in control versus acidotic mice.

Supplemental Table 2. Evaluation of net acid excretion in human urine.

Supplemental Table 3. Evaluation of net acid excretion in Brattleboro rat urine.

Supplemental Table 4. Measurement of the standard for experiments in Supplemental Tables 2 and 3.

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Keywords:

acid-base homeostasis; antidiuretic hormone; distal renal tubular acidosis; intercalated cells; vasopressin V1a receptor; V-ATPase

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